In the last few years, it has been demonstrated by this group and others that rat liver peroxisomes participate in cholesterol synthesis. These unexpected findings raise interesting questions about the characteristics of the proteins involved in peroxisomal cholesterol synthesis, the fate of peroxisome-synthesized cholesterol, and the regulation and function of the peroxisomal system. Aim I of this proposal will focus on the mechanism of the pathway, while aims 2, 3, and 4 deal with the significance and function of this pathway. 1) What parts of the cholesterol biosynthetic pathway are localized within the peroxisomes? Sections of the cholesterol synthesis pathway will be tested in an in vitro system in order to elucidate which parts of the cholesterol biosynthetic pathway are localized within the peroxisomes (in contrast to those that require the presence of cytosolic enzymes). 2) Is cholesterol synthesis defective in cultured Zellweger (a peroxisomal deficiency disease) fibroblast cell lines? A number of biochemical abnormalities all pertaining to peroxisomal metabolism have been described in this disease. The fibroblasts from these patients show the biochemical features of the disease. In studies of peroxisomal diseases significantly low plasma cholesterol levels have been reported. Experiments are designed to measure cholesterol synthesis, ER and peroxisomal HMG-CoA reductase and sterol carrier protein-2 levels in a number of cell lines belonging to different complementation groups. 3) Are peroxisomes involved in cholesterol synthesis in cultured fibroblasts from patients with mevalonate kinase deficiency? Mevalonate kinase deficient cell lines synthesize sterols at a normal rate despite the apparent absence of cytosolic mevalonate kinase activity. Studies are designed to test if these cells contain a novel (peroxisomal) cholesterol synthesis pathway. 4) Is peroxisome-synthesized cholesterol converted to bile acids in vivo? We will test if [14C]-acetyl-CoA produced from [14C]-lignoceric acid (substrate for peroxisomal beta-oxidation) is incorporated into bile acids in vivo under conditions where peroxisomal cholesterol synthesis predominates. Since peroxisomes are essential for life, studies of the peroxisomal pathway of cholesterol biosynthesis may allow us to determine the basis of the organelle defect and the reason for the hypocholesterolemia in peroxisomal disorders. In addition, the study may also provide insights about normal cholesterol and lipoprotein metabolism.